It is valuable for preventive and therapeutical treatment of various diseases to assay clinically specific active substances including hormones, enzymes, antibodies, etc in biologically derived samples such as blood, urine and cerebrospinal fluids and tissues.
Such a clinical assay is conducted as a routine work in medical centers, hospitals and the like. However, conventional methods usually take a much time, and require relatively a large amount of samples and complicated procedures. These are inconvenient for both patients and those who participate in medical practice. Therefore, it has been desired in medical treatments to develop an assay which requires only a small amount of samples and gives accurate results in a short period of time.
As the clinical assay, there have recently widely been used immunoassays, which are based on the specific reaction between two immunologically active substances, (e.g. an antigen and an antibody), one of which is labeled with a detectable marker. Numerous markers are known for use in immunoassay, for example, enzymes, radioisotopes, fluorophores and the like. Among them, enzymes are especially useful for accuracy because of their substrate specificity. In the case of a typical enzyme immunoassay, a test sample is treated with an immunologically active substance (A) (e.g. an antigen or an antibody) which has previously been labeled with an appropriate enzyme, by which an immunologically active substance (B) (e.g. an antigen or an antibody) contained in the test sample is specifically bound with the substance (A), and then, the enzymatic activity in the bound substance is measured by a conventional method, usually by a colorimetry. The reaction is usually carried out in the presence of an appropriate substrate (e.g. which can be colored by the catalytic effect of said enzyme). According to this method, the amount of the substance (B) in the test sample can quantitatively be determined.
It is well known that the enzyme immunoassay includes "heterogeneous immunoassay" (e.g. "ELISA") and "homogeneous immunoassay" (e.g. "EMIT"). The former employs a phase separation procedure as an integral part of the method, while the latter is conducted in a single reaction mixture. ELISA is applicable to a wide range of substances in comparison with other methods. This invention provides an improvement in ELISA.
ELISA can be classified into two assays by the type of the reaction, that is, a competitive reaction and a non-competitive reaction. In the competitive assay, a test sample containing an unknown amount of an antigen (B) is reacted with a known amount of an immobilized antibody (A) in the presence of an enzyme-labeled antigen (B'), wherein the antigen (B) and the enzyme-labeled antigen (B') are competitively reacted with the immobilized antibody. After the reaction, the immobilized antibody (A)-bound enzyme-labeled antigen (A') is separated from the reaction mixture (usually by decantation), and the catalytic (enzymatic) activity of the bound product is measured. In this competitive reaction, the amount of the enzyme-labeled antigen (B') to be bound to the antibody (A) is decreased by the amount of the antigen (B) contained in the test sample, in other words, the amount of the antigen (B) in the test sample is inversely proportional to the amount of enzyme-labeled antigen (B') which was bound to the immobilized antibody. Accordingly, by measuring the enzymatic activity of the bound enzyme-labeled antigen, there can be measured the amount of the antigen (B) contained in the test sample. On the other hand, in the non-competitive immunoassay (e.g. sandwich assay), an immobilized antibody (A) is firstly contacted with a test sample containing an unknown amount of an antigen (B), and the bound sample is contacted with a known amount of an enzyme-labeled antibody (A'). After the reaction, the bound product of the antigen (B)-the enzyme-labeled antibody (A') is separated from the reaction mixture, and the amount of the bound enzyme-labeled antibody is measured on the basis of the catalytic (enzymatic) activity by a conventional method. The amount of the bound enzyme-labeled antibody is proportional to that of antigen (B) in the test sample. Thus, the amount of the antigen in the test sample can quantitatively be determined.
As mentioned above, these enzyme immunoassays employ an antigen (or antibody) immobilized on the surface of a suitable solid carrier. The solid carriers include various kinds of natural or artificial products, such as polyacrylamides, nylons, glass, etc. In general, porous beads carriers are used in ELISA assay. When the beads carrier is used as a solid phase in ELISA, an immobilized substance (e.g. antigen) which is obtained by fixing the protein on the surface of the beads is reacted with an enzyme-labeled substance (e.g. antibody) competitively or non-competitively. To this reaction mixture, any suitable color-producing reagents such as pigments which can be colored by the catalytic effect of the enzyme is added, and colored reagents are extracted from the reaction mixture. Then, the extract is subjected to the spectrophotometric measurement.
However, the conventional ELISA assay using a solid beads carrier has to be conducted in liquid phases and requires troublesome laboratory procedures such as separation and extraction of colored reagents and spectrophotometrical measurements of the extract. Because of such laboratory procedures, this method can not give the desired data rapidly. It is very important to get rapid and correct informations from these assays instantaneously in order to apply the most suitable diagnoses and treatments to each patient. This is especially significant in case of an emergency. Thus, it has been desired to find an improved immunoassay among people who participate in suitable for clinical assay of diseases, particularly for diagnosis of diseases in the early stage, for instance, in diseases participated by antigen-antibody reaction or various infectious diseases.